J. Agric. Food Chem. 2007, 55, 6039−6043
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Isolation of Two Bioactive ent-Kauranoids from the Leaves of Isodon xerophilus ZHI-YING WENG,†,‡,§ SHENG-XIONG HUANG,†,‡ MA-LIN LI,§ YUE-QIN ZENG,⊥ QUAN-BIN HAN,† JOSE LUIS RIOS,*,⊥ AND HAN-DONG SUN*,† State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204, People’s Republic of China; Graduate School of the Chinese Academy of Sciences, Beijing 100039, People’s Republic of China; Yunnan Laboratory of Pharmacology for Natural Products, Kunming Medical College, Kunming 650031, Yunnan, People’s Republic of China; and Department of Pharmacology, University of Valencia, 46100 Burjassot, Spain
Isodon xerophilus has been used as a herbal cold tea for the prevention and treatment of sore throat and inflammation in southernwestern China. A phytochemical study on the ethyl acetate (EtOAc) soluble fraction of I. xerophilus leaves led to the isolation of two new ent-kauranoids, xerophinoids A (1) and B (2), together with 14 known diterpenoids. The structures of xerophinoids A (1) and B (2) were illustrated using spectroscopic methods including 1D and 2D NMR analyses. To study their biological activities, the effects of xerophinoids A (1) and B (2) on nitrite production, tumor necrosis factor (TNF)-R and interleukin (IL)-1β were examined. In addition, xerophinoids A (1) and B (2) also exhibited potent cytotoxicity against several human tumor cell lines (IC50 < 11 µM), but they had no toxicity on human T-lymphocyte (C8166). KEYWORDS: Anti-inflammation; cytotoxicity; herbal tea; Isodon xerophilus; ent-kaurene diterpenoid
INTRODUCTION
MATERIALS AND METHODS
Isodon xerophilus is a perennial plant widely distributed in southern China. Its leaf is traditionally used to prepare healthy herbal beverages, popularly called “liang cha” or “cold tea”. This herbal tea is believed to be a good cold remedy; it helps to treat fever and to reduce the achiness, congestion, and inflammation associated with the common cold. Phytochemical analysis has demonstrated that diterpenoids in Isodon species are one group of the most biologically active compounds against inflammation and cancer (1). We have studied over 50 Isodon species distributed in China, finding about 500 new diterpenoids (2). Among them, total diterpenoids extracted from I. xerophilus are most effective against tumor cells. It has been found that the EtOAc extract of I. xerophilus potently inhibits the growth of various tumor cells including K562, HL-60, HCT, and MKN-28 in vitro (3-7). The objective of the present study was to isolate and identify the structures of two new ent-kauranoids, xerophinoids A (1) and B (2), and to investigate their bioactivity toward tumor cell lines and some inflammatory mediators.
General Experimental Procedures. All melting points were measured on an XRC-1 micro melting point apparatus and are uncorrected. Optical rotations were carried out on a Perkin-Elmer model 241 polarimeter. UV spectra were obtained in a UV 210A spectrometer. IR spectra were measured in a Bio-Rad FTS-135 spectrometer as KBr pellets. 1D and 2D NMR spectra were taken on a Bruker AM-400 or a Bruker DRX-500 NMR spectrometer with TMS as internal standard. Mass spectra were recorded on a VG Auto spec-3000 spectrometer or on a Finnigan MAT 90 instrument. Semipreparative HPLC was performed on an Agilent 1100 liquid chromatograph with a Zorbax SB-C18, 9.4 mm × 25 cm column. Column chromatography was performed on silica gel (200-300 mesh; Qingdao Marine Chemical Inc., Qingdao, People’s Republic of China), Lichroprep RP-18 gel (4063 µm, Merck, Darmstadt, Germany), MCI gel (75-150 µm, Mitsubishi Chemical Corp., Tokyo, Japan), and Sephadex LH-20 (Pharmacia). Plant Material. The leaves of I. xerophilus were collected in Jianshui county of Yunnan Province, People’s Republic of China, in October 2003. The identity of plant material was verified by Prof. Zhong-Wen Lin, and a voucher specimen (KIB 98-11-25 Lin) was deposited in the Herbarium of the Department of Taxonomy, Kunming Institute of Botany, Chinese Academy of Sciences. Extraction and Isolation. The dried and powdered leaves (1.5 kg) were extracted with 70% Me2CO and filtered. The filtrate was concentrated and extracted with EtOAc and n-BuOH successively. The EtOAc layer (50 g) was decolorized on an MCI-gel CHP 20P column (5.0 × 60 cm) with 90% aqueous MeOH and 100% MeOH. The 90% aqueous CH3OH fraction (46 g) was chromatographed over a silica gel column (8 × 120 cm) and eluted in a step gradient manner with
* Corresponding authors [(J. L. R.) e-mail
[email protected] (H.-D.S.) e-mail
[email protected]]. † Kunming Institute of Botany, Chinese Academy of Sciences. ‡ Graduate School of the Chinese Academy of Sciences. § Kunming Medical College. ⊥ University of Valencia.
10.1021/jf070734w CCC: $37.00 © 2007 American Chemical Society Published on Web 06/27/2007
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Table 1. 1H and 13C NMR Data of Xerophinoids A (1) and B (2) (400 and 100 MHz, in C5D5N, J in Hertz) xerophinoid A position 1a 1b 2a 2b 3a 3b 4 5 6 7 8 9 10 11a 11b 12a 12b 13 14 15 16 17a 17b 18 19 20 OAC
δH 1.34 ma 1.01 m 1.23a 1.35a 1.15a 1.25a 1.85 d (13.0) 5.66 d (13.0) 2.60 br s 5.70 br s 2.19 m 2.07 m 3.27 m 4.79 br s 6.23 br s 5.39 br s 1.40 s 1.09 s 10.9 s 1.91 s
OMe a
xerophinoid B δC
δH
δC
35.3
1.98 br d (13.5) 0.85 dt (13.5, 4.0)
30.1
19.3 43.3 35.4 56.9 74.0 204.5 67.0 61.2 55.5 65.9 36.4 45.3 76.0 199.4 149.0 113.3 35.0 21.4 207.0 168.9 21.1
18.5 1.40a 1.17 m 1.34a 4.15 dd (10.1, 6.5) 1.66 dd (12.0, 6.1) 2.53 m 1.32a 2.28 m 1.41 m 3.19 br d (9.4) 5.47 br s 6.24 br s 5.47 br s 1.25 s 1.01 s 5.31 br s 3.44 s
41.5 34.0 59.9 74.8 99.8 62.2 53.1 39.6 19.5 30.8 44.3 73.8 210.0 153.4 119.0 34.2 22.5 103.6 56.3
Overlapped signals.
CHCl3/acetone (1:0 to 0:1, v/v) to yield xerophilusin G (2 g) and fractions I-VII: fraction I (254.7 mg), fraction II (807.2 mg), fraction III (5.2 g), fraction IV (2.8 g), fraction V (1.1 g), fraction VI (691.7 mg), and fraction VII (656.7 mg). Fraction III was chromatographed on a silica gel column (5.0 × 60 cm) with petroleum ether/ Me2CO (8:2-1:1, v/v) to give compound 1 (340 mg), rosthorin A (1 g), and eight fractions. Fractions 3, 4, and 5 (total 2.1 g) were subjected to chromatography over RP-18 (column 3.0 × 40 cm) with MeOH/ H2O(30-100%) to give xerophilusin A (24 mg) and xerophilusin B (19 mg). Fraction 6 (32 mg) was separated by semipreparative HPLC (50% MeOH/H2O) to yield compounds ponicidin (8 mg) and rabdotermin F (3) (12 mg). Fraction IV was chromatographed on an RP-18 column (3.0 × 40 cm) with MeOH/H2O(30-100%), and three fractions were obtained. Fraction 2 (1.2 g) was chromatographed on a silica gel column (3.0 × 30 cm) with petroleum ether/Me2CO (30:1-10:1, v/v) to give xerophilusin H (16 mg), xerophilusin K (10 mg), longikaurin B (31 mg), and 10 fractions. Fraction 4 (102.1 mg) was chromatographed on a silica gel column (1.5 × 25 cm) with cyclohexane/ isopropyl alcohol (50:1-10:1, v/v) to yield compound 2 (32 mg). Fraction 5 (231.4 mg) was also chromatographed on a silica gel column (2 × 25 cm) with cyclohexane/isopropyl alcohol (50:1-10:1, v/v) to obtain macrocalin B (67 mg) and effusanin A (8 mg). Rubescensin C (11 mg) and rabdoternin D (8 mg) were obtained on a semipreparative HPLC (9.4 mm × 25 cm column) with 45% MeOH/10% CH3CN/H2O (3 mL/min) from fraction 5. Fraction 8 (27 mg) was separated in a semipreparative HPLC (45% MeOH/10% CH3CN/H2O) to give rubescensin D (13 mg). Xerophinoid A (1). Compound 1 was named xerophinoid A. Its chemical and physical properties were as follows: white amorphous powder; [R]28 D , -25.52 (c 0.15, CH3OH); UV λmax (MeOH) nm (log ), 238 (3.84); IR (KBr) νmax cm-1, 3467, 2954, 2908, 2848, 1748, 17111705, 1647, 1432, 1371, 1232, 1288, 1049, 1014, 960; 1H and 13C NMR data (see Table 1); positive ESIMS, m/z 427 [M + Na]+; positive HRESIMS [M + Na]+, m/z 427.1734 (calcd for C22H30O8Na [M + Na]+, 427.1732).
Xerophinoid B (2). Compound 2 was named xerophinoid B. Its chemical and physical properties were as follows: white amorphous powder (MeOH); [R]28 D , -20.49 (c 0.26, CH3OH); UV λmax (MeOH) nm (log ), 240 (3.92); IR (KBr) νmax cm-1, 3387, 2948, 2875, 1711, 1643, 1552, 1453, 1369, 1094; 1H and 13C NMR data (see Table 1); positive ESIMS, m/z 401 [M + Na]+; positive HRESIMS [M + Na]+, m/z 401.1943 (calcd for C21H30O6Na [M + Na]+, 401.1940). Cytotoxicity. The cytotoxicity of compounds xerophinoids A and B against murine RAW 264.7 macrophages, rat neutrophils, human leukemia cells (K562, NB4, NB4-R2), solid tumor cells (T-24, Me180, BIU87), rat hematopoietic stem cells (32D), and human T-lymphocyte (C8166) was determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay (3). Cells in a 96-well plate were continuously exposed to different concentrations of compounds (10210-2 µM) for 48 h. The OD of each well was measured on a microplate reader (Bioteck EL-340) at two wavelengths (570 and 630 nm). Nitrite Production in Intact RAW 264.7 Macrophages (8). Murine macrophages RAW 264.7 were cultured in DMEM containing 2 mM L-glutamine, 100 units/mL penicillin, 100 µg/mL streptomycin, and 10% fetal bovine serum. Cells were removed from the tissue culture flask using a cell scraper and resuspended until a final relation of 1 × 106 cells/mL. Nitrite production is assessed as the index of nitric oxide (NO) generation in the induction phase. Macrophages (RAW 264.7) were co-incubated in a 96-well culture plate (200 µL) with 1 µg/mL of lipopolysaccharide (LPS) at 37 °C for 24 h in the presence of test compounds at different concentrations (from 10.0 to 0.05 µM) or vehicle (phosphate-buffered saline). Nitrites were determined in culture supernatant by Griess reagent. Absorbance was measured at 570 nm using a Labsystems Multiskan EX plate reader (Midland, Canada). Results were expressed in absolute absorbance readings; a decrease indicated a reduction in cell viability. Determination of Cytokine Production in RAW 264.7 Macrophages. Cytokine production was determined according to the protocol described by Kuo et al. (9) with modification. RAW 264.7 macrophages (106 cells per well) were co-incubated in a 24-well culture plate (200 µL) with 1 µg/mL of LPS at 37 °C for 3 h. After that, 10 µL of a solution of phytohemagglutinin (PHA, 0.5 mg/mL) was added, and cells incubated at 37 °C during 24 h for interleukin (IL)-1β or 72 h for tumor necrosis factor (TNF)-R, in the presence of test compounds at different concentrations (10.0-0.05 µM). Then, plates were centrifuged at 1200 rpm during 5 min for cell sedimentation, and supernatants were collected and assayed for cytokine production by a specific enzyme immunoassay eBioscience (San Diego, CA), according to the manufacturer’s instructions. RESULTS AND DISCUSSION
Xerophinoid A (1) was obtained as an amorphous powder. The molecular formula C22H30O8 was deduced from the molecular ion peak at m/z 427 [M + Na]+ in ESIMS and NMR data and further confirmed by the HRESIMS (m/z 427.1732 [M + Na]+). The IR spectrum showed absorption bands due to hydroxyl groups (3467 cm-1), carbonyl groups (1748 and 1711 cm-1), and a CdC double band (1647 cm-1). The 1H NMR spectrum displayed three tertiary methyl signals (δH 1.09, 1.40, and 1.90, each 3H, s), two olefin signals (δH 5.39 and 6.23, each 1H, br s), and an aldehyde signal (δH 10.9, s). The 13C and DEPT NMR spectra revealed a total of 22 carbon signals including 3 methyls, 5 methylenes, 6 methines including 3 oxymethines, 1 double bond, 4 carbonyl groups including 1 aldehyde, and 3 nonoxygenated quaternary carbons. These NMR data together with the molecular formula suggested that xerophinoid A has four rings. Considering the NMR data and the structural types of diterpenoids isolated from I. xerophilus previously, xerophinoid A was assigned tentatively as an entkauranoid, which was confirmed by the measurement of extensive 2D NMR data (Figures 1 and 2). In the 1H-1H COSY and HSQC spectra of xerophinoid A, the proton spin systems H-1/H-2/H-3 and H-9/H-11/H-12/H-13/H-14 were deduced
Characterization of Xerophinoids A and B in I. xerophilus
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Figure 1. Chemical structures of xerophinoid A (1), xerophinoid B (2), and rabdoternin F (3).
Figure 2. Selected COSY and HMBC correlations of xerophinoid A (1).
Figure 3. Key ROESY correlations of xerophinoid A (1).
(Figures 1 and 2). Observations of the HMBC correlations of H-5 (δH 1.85 d, J ) 13.0 Hz) with C-1, C-6, C-7, C-9, C-10, C-18, and C-19, H-9 (δH 2.60, br s) with C-8, C-10, C-12, C-14, C-15, and C-20, and H2-17 (δH 6.23 and 5.39, each 1H, br s) with C-15, C-16, and C-13 led us to establish the ent-kauranoid skeleton. According to the correlations in the HMBC and COSY spectra of xerophinoid A, the acetoxyl group was placed at C-11, the aldehyde was placed at C-20, and two ketone groups were placed at C-7 and C-15, respectively. The relative stereochemistry of xerophinoid A was deduced from coupling constants and correlations observed in ROESY spectrum as shown in a computer-generated 3D drawing (Figure 3). The trans coupling constant between H-6 and H-5 and ROESY correlations of H-6 with Me-19 and H-20 showed the β-orientation of OH-6. The β-orientation for OH-14 was suggested from the ROESY cross-peak of H-14 with H-20 and H-12R. The dihedral angles between H-11 and H-9 were close to 90° and 45°, when the 11-OAc group was in an R- or β-orientation, respectively. Therefore, the coupling constants between H-9 and H-11 could be used in the determination of the relative configuration of C-11 of xerophinoid A. The singlet signal of H-9 indicated that the 11-OAc group was in a β-orientation, which was also supported by the ROESY cor-
relations of H-11 with H-1R. The absolute configuration of xerophinoid A was not determined experimently, although it was assumed to belong to the ent-series of kaurane. This was the most commonly observed configuration for this class of diterpenoids and appeared to be favored on biogenetic grounds in Isodon species. On this basis, xerophinoid A was characterized as 6β,14β-dihydroxy-11β-acetoxy-ent-kaur-7,15-dioxo-20al-16-ene. Xerophinoid B (2) was assigned the molecular formula C21H30O6, as deduced from the positive HRESIMS (m/z 401.1936 [M + Na]+). Comparison of the spectroscopic data of xerophinoid B with those of rabdoternin F (3) revealed that they were quite similar except for the moiety at C-1 (Figure 1). Observation of the presence of a methylene carbon (δC 30.1, t) and the absence of an oxymethine carbon in the 1H and 13C NMR spectra of xerophinoid B showed that a methylene carbon at C-1 position was evident for xerophinoid B instead of a hydroxyl group at the same position in rabdoternin F (3). Moreover, the correlations in the ROESY spectrum of xerophinoid B indicated that the corresponding substituents in xerophinoid B had the same orientaton as those in rabdoternin F (3). Thus, compound xerophinoid B was determined as (20S)-6β,7β,14β-trihydroxy-20-methoxy-7,20-epoxy-ent-kaur-16-en-15one. The structures of other compounds isolated were already known, namely, rabdoternin F (10), xerophilusin A (3), xerophilusin B (3), macrocalin B (11), xerophilusin G (12), xerophilusin H (12), xerophilusin K (5), ponicidin (6), rabdoternin D (5), longikaurin B (5), rosthoriin A (5), effusanin A (13), rubescensin C (14), and rubescensin D (15). The structural identification was made by comparing their spectroscopic data with those in the literature. Xerophinoids A and B were examined for their cytotoxicity against seven kinds of human tumor cells, human T-lymphocyte (C8166), and rat hematopoietic stem cells (32D). As shown in Table 2, it is noticeable that two new diterpenoids showing cytotoxicity against tested tumor cells except for C8166, T-24,QGY-7202, and BIU-87 cells (IC50 < 11 µM) indicate their broad cytotoxic activities. The IC50 values against rat hematopoietic stem cells were all 25 µM, indicating their side effects should be major in toxicity to hematopoietic stem cells. Xerophinoid B exhibited greater inhibition on K562, NB4, NB4R2, Me-180, BIU-87, and 32D cells but weaker inhibition on T-24 and QGY-7202 than xerophinoid A. They showed selective cytotoxicity on different human tumor cell lines, which might be due to their different substituents at C-20. In addition, the toxicity of xerophinoid A against murine RAW 264.7 macrophages and rat polymorphonuclear leukocytes was >10 µM, whereas thye toxicity of xerophinoid B was >1 µM against both cell lines.
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Table 2. Cytotoxicity of Xerophinoids A (1) and B (2) on Different Cell
Lines IC50a (µM) cell line
xerophinoid A
xerophinoid B
K562 NB4 NB4-R2 T24 Me180 BIU87 QGY-7202 32D C8166
4.48 ± 1.40 10.09 ± 2.06 3.98 ± 0.87 24.59 ± 3.67 5.29 ± 1.34 40.27 ± 1.94 19.52 ± 1.81 4.90 ± 1.31 >25
0.32 ± 0.13 3.73 ± 0.67 3.06 ± 0.90 45.98 ± 2.31 3.46 ± 0.46 32.22 ± 3.16 27.06 ± 2.59 3.99 ± 0.91 >25
a IC was calculated from the dose response linear regression plots (100− 50 0.01 µM). The results were expressed as mean ± SEM, n ) 3.
Table 3. Inhibitory Effect of Xerophinoids A (1) and B (2) on Production of NO, TNF-R, and IL-β in LPS-Stimulated Cells and in PHA-Stimulated Cellsa NO
TNF-R
IL-β
compound
%I
IC50
%I
IC50
%I
IC50
xerophinoids A xerophinoids B dexamethasone
98** 100** 65**
2.10 0.24
41** 57** 91**
>10 >1
100** 100** 89**
